The invention discloses a method for separating and purifying cardiac myocytes, and relates to the technical field of biological cell culture. The method comprises the steps of S1, adding prepared coating liquid into a six-well plate for performing incubation; S2, adding a myocardial buffer solution into a petri dish, and rinsing a heart placed in the petri dish containing the myocardial buffer solution; S3, cutting out heart tissues and putting the cut heart tissues into a centrifuge tube containing the myocardial buffer solution; S4, adding 0.05% of type II collagenase into the centrifuge tube, carrying out shake cultivation, and discarding supernatant; S5, adding 0.1% of type II collagenase into the centrifuge tube treated in the step S4, carrying out shake cultivation, taking supernatant to put into a centrifuge tube filled with serum, and then evenly mixing; S6, after the supernatant is evenly mixed, then filtering, and centrifuging; after centrifuging is finished, resuspending byusing the myocardial buffer solution, and adding cell suspension into the centrifuge tube; S7, slowly adding the cell suspension into a prepared density gradient solution, and performing centrifugation; S8, placing the separated intermediate layer in a centrifuge tube, centrifuging, then resuspending, recording data, and then adding the product into the six-well plate treated in the step S1 for culturing.